Dynamic BH3 profiling identifies pro-apoptotic drug combinations for the treatment of malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) has relatively ineffective first/second-line therapy for advanced disease and only 18% five-year survival for early disease. Drug-induced mitochondrial priming measured by dynamic BH3 profiling identifies efficacious drugs in multiple disease settings. We use high throughput dynamic BH3 profiling (HTDBP) to identify drug combinations that prime primary MPM cells derived from patient tumors, which also prime patient derived xenograft (PDX) models. A navitoclax (BCL-xL/BCL-2/BCL-w antagonist) and AZD8055 (mTORC1/2 inhibitor) combination demonstrates efficacy in vivo in an MPM PDX model, validating HTDBP as an approach to identify efficacious drug combinations. Mechanistic investigation reveals AZD8055 treatment decreases MCL-1 protein levels, increases BIM protein levels, and increases MPM mitochondrial dependence on BCL-xL, which is exploited by navitoclax. Navitoclax treatment increases dependency on MCL-1 and increases BIM protein levels. These findings demonstrate that HTDBP can be used as a functional precision medicine tool to rationally construct combination drug regimens in MPM and other cancers.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. No sample size for in vitro experiments was determine, so we used n=3 independent experiments. Therefore, all in vitro cell line experiments were done at least in 3 biological replicates and each independent experiment, where possible, was also carried out using technical repeats. MPM patient samples were precious with only one sample per patient because you receive the sample after the patient has surgery for tumor resection. Due to this limited patient sample size we did all these experiments in technical duplicates. We wanted to treat the MPM PDX tumor samples the same as we did for the patient so for each tumor harvested we carried out CROCS HTDBP in technical duplicates. We implanted MPM PDX tumors in the right and left flank of two mice, so each model had the chance to grow up to 4 tumors per model. However, not all sites grew tumors. We did not use a statistical method to decide sample size for animal studies but keeping the 3R's in mind we did not want to use too few mice and jeopardize reliability of the results or use too many mice, because the risk making a false positive conclusion increases. With that in mind and with our previous animal study experience we decided to use 7-9 mice per arm for the in vivo efficacy study. Three mice for the pharmacodynamic study (iBH3 profiling and Western blotting analysis).
All animals that were implanted with CPDM_0011x tumors were enrolled in the efficacy study or pharmacodynamic study (iBH3 profiling and Western blotting analysis). Because the tumor take rate was 100% for this model, no animals were excluded from this study. The take rate for CPDM_0106x tumor growth and CPDM_0184x tumor growth was 50%. There were no exclusion criteria for in vitro cell line data or MPM patient sample ex vivo data. All data was included in the manuscript.
All in vitro experiments were carried out in 3 biological replicates. All replicates were successful and we included all replicates of experiments in our data analysis. CROCS HTDBP was carried out in technical duplicates because patient samples are precious.
For in vivo efficacy studies mice bearing CPDM_0011x tumors (Figure 4a-d), measuring between 150-250 mm3 were randomized, using the deterministic method, into 5 groups of 7-9 mice in a rolling recruitment method. This method was also used for in vivo pharmacodynamic studies when tumors reached~800 mm3 (Figure 4e-f) . No randomization method was needed for data generated else where in the paper because we used all available patient tumors and MPM PDX tumors for ex vivo CROCS HT-DBP screen.
We were blinded to any identifying information from patients who signed up for the study.